Fig. 6: Subdiffraction image analysis of fluoresence polarisation microscopy (FPM) and frame-separated fluorescence excitation polarization angle narrowing polarization microscopy (FrExPAN) data.

a Setup for fluorescence polarization microscopy by rotation of the excitation polarization (FPM). b Diffraction-limited image of single molecules and c false color version of the diffraction-limited image, color-coded based on the different orientations. d Results of the deconvolution with the SPEED algorithm to locate the molecules at sub-diffraction-limited resolution (single molecule Data previously published in Hafi, N. et al., Nature methods 13, 8–9 (2016)³⁴). e Two selected phase ranges from this analysis, separating molecules in different orientation ranges. f–t Shows different examples of linear actin filaments on a coverslip labeled with phalloidin-Atto 590 (averaged raw data previously published in Hafi, N. et al., Nature methods 13, 8–9 (2016)³⁴)³⁵. f Averaged raw data with (k, p) orientation color-coded images of the diffraction-limited images. g, l, q Deconvolved images based on the entire signal and h, m, r deconvolved images based on only the modulating part (A in eq. 1,2) and i, j, n, o, s, t selected phase/orientation ranges using the ALPA algorithm (for details see methods section). u–ad FPM results of data from a double membrane-anchored dt-p-rsGreenF-F living HeLa cell without second FrExPAN pulse (NoExpan). u, z Averaged diffraction-limited raw data. v, aa FPM data deconvolved and orientation color-coded results after 500 iterations with Richardson–Lucy. w–y, ab–ad FPM data deconvolved and orientation color-coded images after 500 iterations with SPEED algorithm and with selected phase/orientation ranges (for details see text). ae Set-up for frame-separated fluorescence excitation polarization angle narrowing polarization microscopy (FrExPAN, for details see Fig. 5 and corresponding text). af–an Results of the FrExPAN data obtained under identical conditions as in (u–ad) but with second ExPAN pulse. aj Averaged diffraction-limited raw data. af, ak Deconvolved images after 500 iterations with a Richardson–Lucy algorithm. ag–ai, al–an Deconvolved images after 500 iterations with the SPEED algorithm. Scale bars b–e 1 µm, f–t 0.25 µm, and u–an 2 µm. (The corresponding modulation signals are shown in Supplementary Movies 3, 4) For details, see text.